Vibrio cholerae causes the fatal epidemic diarrheal disease cholera. The expression of its primary virulence factors, toxin-coregulated pilus and cholera toxin, occurs via a transcriptional cascade involving several activator proteins and serves as a paradigm for the regulation of bacterial virulence. AphA and AphB initiate the expression of the cascade by a novel interaction at the tcpPH promoter. AphA is a member of a new regulator family and AphB is a LysR-type activator, one of the largest transcriptional regulatory families. Once expressed, cooperation between TcpP/TcpH and the homologous transmembrane activators ToxR/ToxS activates the toxT promoter. ToxT, an AraC-type regulator, then directly activates the promoters of the primary virulence factors in a fatty acid dependent manner. Transcriptional activation at these various promoters occurs only in response to certain environmental stimuli. One such stimulus, cell density, influences the virulence cascade through the quorum sensing system regulator HapR which represses the expression of the aphA promoter. The long term goals of this proposal are to understand the molecular basis of virulence gene regulation so as to facilitate the development of better strategies to prevent and cure bacterial diseases. Achieving these goals requires an understanding of how the specific regulatory proteins function at their promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. Through a collaborative effort involving laboratories with expertise in structural biology, virulence gene regulation, and pathogenesis, we have solved crystal structures of AphA, AphB, HapR, and ToxT. This proposal will build upon this structural data, as well as our functional results, in ordr to continue to elucidate the detailed mechanisms required for regulation of the V. cholerae virulence genes. In Aim 1, we propose to determine the crystal structures of AphA, AphB, and HapR in complex with their respective DNA binding sites, allowing us to observe the structural changes that take place upon DNA binding. In Aim 2, we plan to characterize the ligand binding pockets of AphB and HapR. As the natural ligands for these proteins are not known, we plan to identify small molecule ligands for HapR and AphB, and then visualize the structural changes induced in the proteins by ligand binding. Aim 3 carries on our investigation of the mechanism by which fatty acid binding regulates the activity of ToxT, the master regulator of virulence gene expression in V. cholerae. In addition to crystallography, we will utilize structure based site directed mutagenesis, biochemical activity assays, biophysical characterization assays, spectroscopic characterization of binding and conformational change, and computational and NMR based methods for identifying ligands. These studies will greatly clarify the mechanistic and structural roles of proteins involved in the regulation of bacterial virulence gene expression. Such knowledge will facilitate the identification of molecules interfering with regulatory cascades, and could lead to the development of novel therapeutics.